Fluorescence polarization technique can specifically detect nucleotide sequences by hybridization without separation procedures. We applied the technique to the detection of the Shiga toxin genes (stx1andstx2) in enterohemorragicEscherichia coli(STEC). STEC possessesstx1 and/orstx2 and using MK primers, the amplification ofstx1 and/orstx2 can be performed universally, but the band sizes for both genes are almost identical; thus the genotype is hard to determine using electrophoresis. To identify stx genotype, conventional PCR combined with electrophoresis requires a second amplification process with more than 25 temperature cycles using other genotype-specific primer pairs. However, using the newly developed method only a few temperature cycles of the asymmetric PCR using the same MK primers and two different probes forstx1andstx2 were needed. The incubation time for probe hybridization was 10 minutes, and the detection time for fluorescence polarization was within about 1 minute per sample. Thus, we considered that the detection method using fluorescence polarization technique was a rapid and reliable method for detectingstx1and/orstx2.
We investigated the usefulness of a rapid screening method for the detection of VTEC andSalmonellafrom swab specimens of flayed carcasses by detecting the Vero toxin (VT) andSalmonella invAgenes by PCR using multiplex primer sets (MK and SIN primer sets) for the meat inspection at slaughterhouse. Thirteen strains of VTEC and 3 strains ofSalmonellawere examined in the presence of other bacteria (non-VTEC and non-Salmonella) concomitantly present in the swab specimens. The multiplex PCR could detect VT andinuAgenes at a concentration of 2.0×104cfu/ml and 2.1×103cfu/ml respective1y, even in the presence of other bacteria at a concentration of 109cfu/ml in the broth suspension. Brief shaking-incubation of the swab specimens at 36°Cfor 8 hrs in Tryptic Soy Broth (TSB), during which period both VTEC andSalmonella, if present, grew to 106.6-108.4cfu/ml, enhanced the detection rate of the multiplex PCR test. Cultivation of the swab specimens in either EEM or N-mEC media showed restricted growth, and subsequent lower detection rates compared with that in TSB. With the combination of brief shaking-culture in TSB and the multiplex PCR, we could detect as little as 1-10cfu/ml of VTEC andSalmonellapresent in the swab specimens. This method can shorten the time and reduce the number of staff needed to perform meat inspection.